Screening of Antiglaucoma, Antidiabetic, Anti-Alzheimer, and Antioxidant Activities of Astragalus alopecurus Pall—Analysis of Phenolics Profiles by LC-MS/MS

Astragalus species are traditionally used for diabetes, ulcers, leukemia, wounds, stomachaches, sore throats, abdominal pain, and toothaches. Although the preventive effects of Astragalus species against diseases are known, there is no record of the therapeutic effects of Astragalus alopecurus. In this study, we aimed to evaluate the in vitro antiglaucoma, antidiabetic, anti-Alzheimer’s disease, and antioxidant activities of the methanolic (MEAA) and water (WEAA) extracts of the aerial part of A. alopecurus. Additionally, its phenolic compound profiles were analyzed by liquid chromatography–tandem mass spectrometry (LC–MS/MS). MEAA and WEAA were evaluated for their inhibition ability on α-glycosidase, α-amylase, acetylcholinesterase (AChE), and human carbonic anhydrase II (hCA II) enzymes. The phenolic compounds of MEAA were analyzed by LC-MS/MS. Furthermore, total phenolic and flavonoid contents were determined. In this context, the antioxidant activity was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), N,N-dimethyl-p-phenylene diamine (DMPD), ferric reducing antioxidant power (FRAP), cupric ions (Cu2+) reducing antioxidant capacity (CUPRAC), ferric ions (Fe3+) reducing, and ferrous ions (Fe2+) chelating methods. MEAA and WEAA had IC50 values of 9.07 and 2.24 μg/mL for α-glycosidase, 693.15 and 346.58 μg/mL for α-amylase, 1.99 and 2.45 μg/mL for AChE, and 147.7 and 171.7 μg/mL for hCA II. While the total phenolic amounts in MEAA and WEAA were 16.00 and 18.50 μg gallic acid equivalent (GAE)/mg extract, the total flavonoid contents in both extracts were calculated as 66.23 and 33.115 μg quercetin equivalent (QE)/mg, respectively. MEAA and WEAA showed, respectively, variable activities on DPPH radical scavenging (IC50: 99.02 and 115.53 μg/mL), ABTS radical scavenging (IC50: 32.21 and 30.22 µg/mL), DMPD radical scavenging (IC50: 231.05 and 65.22 μg/mL), and Fe2+ chelating (IC50: 46.21 and 33.01 μg/mL). MEAA and WEAA reducing abilities were, respectively, Fe3+ reducing (λ700: 0.308 and 0.284), FRAP (λ593: 0.284 and 0.284), and CUPRAC (λ450: 0.163 and 0.137). A total of 35 phenolics were scanned, and 10 phenolic compounds were determined by LC-MS/MS analysis. LC-MS/MS revealed that MEAA mainly contained isorhamnetin, fumaric acid, and rosmarinic acid derivatives. This is the first report indicating that MEAA and WEAA have α-glycosidase, α-amylase, AChE, hCA II inhibition abilities, and antioxidant activities. These results demonstrate the potential of Astragalus species through antioxidant properties and enzyme inhibitor ability traditionally used in medicine. This work provides the foundation for further research into the establishment of novel therapeutics for diabetes, glaucoma, and Alzheimer’s disease.

IOP. For this purpose, β-blockers, topical prostaglandins, CA inhibitors (CAIs), or their combinations are the most commonly applied methods to date [47].
Diabetes mellitus (DM) is a common and multifactorial metabolic disease characterized by hyperglycemia due to insulin deficiency or insulin resistance [51]. According to a classification made by the American Diabetes Association (ADA), diabetes types are classified into four general categories: The first is insulin-dependent Type-1 DM (T1DM), the second is insulin-dependent Type-2 DM (T2DM), the third is neonatal diabetes, and the last is gestational diabetes [52]. Non-insulin-dependent T2DM is the most common type of diabetes and accounts for approximately 90-95% of all diabetes cases [53]. According to the World Health Organization's data, T2DM continues to be the most common and fastest rising health problem in developing countries [54]. In T2DM, long-term high glucose levels trigger the deterioration of cellular functions, especially inflammatory and oxidative stress, leading to the development of serious chronic diabetic complications such as neuropathy, cataracts, and atherosclerosis [55]. One of the therapeutic approaches to reducing hyperglycemia is inhibition of α-glycosidase and α-amylase, which break down α-1,4 glycosidic bonds from the non-reducing ends of oligosaccharides and polysaccharides, allowing them to be absorbed by the small intestine and enter the bloodstream [56]. α-Glycosidase inhibitors competitively inhibit intestinal α-glycosidase, thereby delaying or decreasing carbohydrate absorption in the small intestine. The use of α-glycosidase inhibitors is widely used to treat diabetes, particularly T2DM. Therefore, the most important features of the ideal antidiabetic agent are that it is of natural origin, has a hypoglycemic effect, and has the ability to prevent long-term diabetic complications [53,57].
In this study, we aimed to determine the antiglaucoma, antidiabetic, anticholinergic, antioxidant, and other activities of methanol (MEAA) and water (WEAA) extracts of the aerial parts of A. alopecurus. For this purpose, the possible inhibitory effects of MEAA and WEAA toward AChE, hCA II, α-amylase, and α-glycosidase enzymes were determined. Another goal of this study was to investigate the antioxidant ability of both extracts with different bioanalytical methods, including potassium ferricyanide reduction, Fe 3+ -2,3,5-Triphenyltetrazolium chloride (TPTZ) reduction (FRAP), copper ion (Cu 2+ ) reducing capacity (CUPRAC), DPPH˙, and ABTS + scavenging and metal chelating. Additionally, the total phenolic and flavonoid contents of MEAA and WEAA extracts were also defined. The polyphenolic analysis of MEAA was quantitatively determined by LC-MS/MS. Another goal of this study was to identify the possible inhibitory effects of MEAA and WEAA on AChE, hCA II, α-amylase, and α-glycosidase metabolic enzymes.

Total Phenolic and Flavonoid Contents
Total phenolic contents in MEAA and WEAA were calculated using standard gallic acid calibration curves by the Folin-Ciocalteu reagent (r 2 : 0.9983) [58]. The quantities of phenolics in MEAA and WEAA were determined using the standard gallic acid equation and found to be 16.00 and 18.50 µg GAE/mg extract, respectively. Furthermore, flavonoids are one of the most abundant secondary metabolites in medicinal plants. It was determined that A. alopecurus contains 66.23 and 33.115 µg QE/mg flavonoid in methanol and water extracts, respectively.

Polyphenolic Analysis by LC-MS/MS
Phenolic compounds are bioactive secondary metabolites found in plants with potential beneficial effects on human health [59]. The LC-MS/MS method is a widely used technique for the analysis of phenolic compounds found in plants. Product ions are produced by tandem mass spectrometry, which allows the characterization of compounds in a given sample. LC-MS/MS is both a powerful and accurate technique for the qualitative and quantitative analysis of phenolic compounds due to its method versatility [60]. In this study, 35 of all known phenolic compounds that have been characterized using the

Antioxidant Results
The reducing activity of MEAA and WEAA was evaluated by measuring their ability to reduce Fe 3+ to Fe 2+ . Various electron-donating functional groups, such as -OH, -SH, and -COOH belonging to the compounds found in plant extracts, are of great importance to The analytical method was validated for linearity, intra-and inter-day precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ). Linearity was demonstrated by linear calibration curves obtained by plotting the peak area versus the concentration of each phenolic compound. The method showed good linearity in the concentration range of 100-2000 µg/L for 30 phenolic compounds, 250-2000 µg/L for 4-OH-benzoic acid and caffeic acid, and 100-1500 µg/L for apigenin, naringenin, and galangin. Intra-and inter-day precision and accuracy were determined by analyzing three replicates of quality control (QC) samples for each phenolic compound on a single day and on three separate days, respectively. The QC samples were the 100, 750, and 1500 µg/L concentrations, except for 4-OH-benzoic acid and caffeic acid (250, 750, and 1500 µg/L) for all phenolic compounds. Precision and accuracy were defined as relative standard deviation (RSD) and relative error (RE), respectively. According to the analyzed results of each phenolic compound, the intra-and inter-day RSD% and RE% were less than 2.48% and ±1.52 at the QC concentrations, respectively. The sensitivity of the method was determined with LOD and LOQ defined as 3SDy/x and 10SDy/x, respectively, where SDy is the standard deviation of the y-intercepts and x is the slope of the calibration curves for each phenolic compound. The highest LOD/LOQ values were obtained for epigallocatechin gallate (1.98/6.6 µg/L) and the lowest for naringin (0.33/1.08 µg/L). The LOD/LOQ values for all other phenolic compounds were within these ranges.
The phenolic compounds in MEAA were determined according to the MS spectra and the retention times of the reference standards. Each sample had two measurements. According to the results, 10 phenolic compounds: fumaric acid, chlorogenic acid, 4-OH-benzoic acid, ellagic acid, p-coumaric acid, rosmarinic acid, luteolin, quercetin, naringenin, and isorhamnetin were identified in MEAA in Table 1. Quantitative analysis of the identified phenolic compounds in MEAA was determined by using calibration curves with seven concentration levels for each analyte, and each level was analyzed in triplicate. When they were ordered from the highest amount to the lowest, isorhamnetin (1489.  (Table 1).

Antioxidant Results
The reducing activity of MEAA and WEAA was evaluated by measuring their ability to reduce Fe 3+ to Fe 2+ . Various electron-donating functional groups, such as -OH, -SH, and -COOH belonging to the compounds found in plant extracts, are of great importance to the reducing capacity [61]. As shown in Figure 3, MEAA and WEAA show inferior reducing power compared to the standards when utilizing the potassium ferricyanide reduction technique. The Oyaizu method [41] was used to explore the Fe 3+ -Fe 2+ transition in order to determine the reductive capacity of MEAA and WEAA, which displayed high reducing activity at various concentrations (15-45 µg/mL). With rising sample concentrations, the reducing power of Trolox, BHA, BHT, α-tocopherol, MEAA, and WEAA continuously rose. The sequence of the standard compounds and extracts reducing abilities was BHT (λ 700 : 2.018) > α-Tocopherol (λ 700 : 1.895) > Trolox (λ 700 : 1.545) > BHA (λ 700 : 1.257) > MEAA (λ 700 : 0.308) > WEAA (λ 700 : 0.284). The outcomes show that plant extracts have the ability to donate electrons to stable compounds, neutralizing free radicals ( Figure 3A and Table 2).

Enzyme Inhibition Results
The antidiabetic activity of MEAA and WEAA was assessed using α-amylase and α-glycosidase inhibition assays in the study. The findings are shown in Table 4. MEAA and WEAA had IC 50 values for α-glycosidase of 9.07 µg/mL (r 2 : 0.9775) and 2.24 µg/mL (r 2 : 0.9155), respectively. This value was determined to be 693.15 µg/mL (r 2 : 0.9677) and 346.58 µg/mL (r 2 : 0.9856), respectively, for α-amylase (Table 4). Acarbose, a common antidiabetic medication, was utilized as a standard reference [62]. According to the findings, methanol and water extracts inhibited both enzymes at a level proximate to that of the standard antidiabetic drug. The results in Table 4 show that aqueous extract inhibited the α-glycosidase and α-amylase enzymes better than methanol extract. In future studies, iso-lation studies on these two extracts will allow for obtaining the pure substances responsible for the effect. Table 4. The inhibition values of MEAA and WEAA against α-glycosidase, α-amylase, acetylcholinesterase (AChE), and carbonic anhydrase II (CA II) enzymes. Acetazolamide is a standard inhibitor of carbonic anhydrase II (CA II) isoenzyme inhibition. Tacrine is a positive control for acetylcholinesterase (AChE) inhibition. Acarbose is a positive control for α-glycosidase and α-amylase enzyme inhibition. The inhibitory effects of MEAA and WEAA on the AChE enzymes associated with AD in different doses were examined, and IC 50 values were obtained. The results in Table 4 show that methanol extract inhibits the AChE enzyme better than aqueous extract. In our study, the results of the effects of MEAA and WEAA on AChE inhibition were evaluated. According to the findings, it was determined that MEAA and WEAA had IC 50 values of 1.99 µg/mL (r 2 : 0.9923) and 2.45 µg/mL (r 2 : 0.9930) for AChE, respectively. In addition, plant extracts were found to have a lower inhibitory activity than standard inhibitory tacrine (IC 50 0.0246 µg/mL; r 2 :0.9706). In the current study, MEAA and WEAA inhibited hCA II isoenzyme with IC 50 values of 147.7 µg/mL (r 2 : 0.9804) and 171.7 µg/mL (r 2 : 0.9671), respectively.

Inhibitors
Additionally, cytosolic and dominant CA II isoenzymes are associated with a number of disorders, such as glaucoma, osteoporosis, and renal tubular acidosis. The IC 50 values of MEAA and WEAA towards CA isoenzymes were found to be 147.7 µg/mL (r 2 : 0.9804) and 171.7 µg/mL (r 2 : 0.9671), respectively (Table 4). On the other hand, acetazolamide (AZA), which is clinically used as a control for the inhibition of CA isoenzymes, displayed an IC 50 value of 8.37 µg/mL (r 2 : 0.9825).
Compared with these studies, our study's sample (A. alopecurus) showed differences in phenolic compounds found by LC-MS/MS analysis. The differences in observed phenolic compounds can be attributed to many factors, such as age, variety difference, growing medium, method of harvesting, and more. However, this is consistent with the literature, as fumaric acid, isorhamnetin, chlorogenic acid, rosmarinic acid, and other phenolics make up a significant part of the phenolic compounds found within Astragalus species. This study provided the first important finding regarding the high concentration of isorhamnetin, which has extensive pharmacological activities, including cardiovascular and cerebrovascular protection, anti-tumor, anti-inflammatory, anti-oxidation, organ protection, prevention of obesity, etc. [67]. Other findings were the presence of high levels of fumaric acid, which is used as a food additive and as a food acidity regulator due to its antimicrobial, antityrosinase, and antioxidant properties, and high levels of rosmarinic acid, which is used to extend the shelf life and improve the quality of foods [70].
In a study, the phenolic contents of the aerial part and root extract of A. dumanii were detected as 13.23 and 5.31 mg GAE/g, respectively, and the flavonoid contents were found to be 7.93 and 8.26 mg QE/g [17], respectively. The total phenolic and flavonoid contents in A. brachycalyx ethanol extracts were found to be 23.182 µg GAE/mg and 4.672 µg QE/mg, respectively [16]. The total phenolic and flavonoid contents in the various extracts of A. lagurus were found to be 20.34-20.72 mg GAEs/g and 19.58-31.10 mg REs/g, respectively [71]. In a study, the total phenolics and flavonoids in the methanol extract of A. squarrosus were found to be 23.3 mg GAEs/g and 26.0 mg QE/g, respectively [72]. In another study, the total phenolics and flavonoids in the methanol extracts of the stem parts of A. diphtherites and A. gymnalopecias were found to be 76.1 ± 0.9, 54.66 ± 2.3 µg GAE/mg and 39.31 ± 0.2, 36.81 ± 0.3 µg QE/mg, respectively. The following results were reported in the study conducted by Albayrak and Kaya [73], in which the phenolic content and antioxidant activity of four Astragalus species (A. gummifer, A. microcephalus, A. talasseu, and A. acmophyllus) were determined: The yields of the plants are between 9.78 and 16.38. Total phenolic contents are between 5.49 and 13.49 mg GAE/g extract. Total flavonoid contents are between 0.76 and 2.19 mg QE/g extract. DPPH IC 50 values are between 86.67 and 253.88 µg/mL. When the percent inhibition values of the iron chelating activity of the extracts were measured at 5 mg/mL, the percent inhibition value of standard EDTA was 99.45%, while the extracts ranged from 43.88% to 68.35%. In addition, the amounts of chlorogenic acid, epicatechin, catechin hydrated, rutin, quercetin, kaempferol, syringic acid, cinnamic acid, and ferulic acid of Astragalus species were determined by LC-MS/MS. In the LC-MS/MS analysis of the extracts, it was reported that the main component was ferulic acid (1123.9 ppm), followed by syringic acid (735.18 ppm) and cinnamic acid (558 ppm), and the least abundant compound was quercetin (293.5 ppm) [73].
It was reported that methanol, ethyl acetate, butanol, and aqueous extracts made from the aerial parts of A. bombycinus had low DPPH free radical scavenging activity. Inhibition values at the dose of 0.1 g/mL were found to be 12.2, 11.5, 8.5, and 8.2 for the extracts, respectively [74]. The IC 50 value for the A. globosus methanolic extract is 196.4. For the positive control, butylated hydroxytoluene, this value is 19.8 µg/mL. In the β-carotene/linoleic acid system, A. globosus methanolic extract has 35.9% activity, while hexane/dichloromethane extract has 48.7% activity [75]. The n-butanol extract of A. monspessulanus' aerial parts was reported as 2.09 g/mL [76]. It has been reported that the IC 50 values of DPPH radical scavenging activity of ethanol extracts obtained from the aerial and root parts of A. dumanii are 1398 and 1009 µg/mL, respectively. In addition, the IC 50 values of ABTS radical scavenging activity were reported as 1.18 and 82.25 µg/mL, respectively [17].
In the study conducted by Albayrak and Kaya [73], the antioxidant activity of four Astragalus species was examined. Ferric ions (Fe 3+ ) reducing the capacity of the extracts are between 0.60 and 4.25 mM/L. When the reducing power of ferric ions (Fe 2+ ) is measured, the absorbance value of standard BHT is 2.249 and that of extracts is between 1.118 and 2.172. When cupric absorbance values are measured at 1 mg/mL, the absorbance value of standard Trolox is 2.85, while the extracts are between 0.32 and 0.83 [77]. In another study, it was reported that A. lagurus water extract had a reducing power of 73.98 mg TEs/g for CUPRAC and 53.49 mg TEs/g for FRAP [71].
Experimental studies support the use of natural products as a source of antioxidants against neurodegeneration [78]. According to the literature review, it was found that the AChE inhibition effects of ether extracts of A. leporinus, A. distinctissimus, and A. schizopterus plants were determined as follows: 46.96 ± 4.06, 54.71 ± 0.09, and 22.01 ± 0.07%, respectively. In the discovery of antidiabetic compounds with fewer side effects, in vitro experiments may be preferred, as in vivo experiments involve more expensive and ethical responsibilities. When the literature was searched, it was observed that A. brachycalyx ethanol extract showed IC 50 values of 0.620 µg/mL on α-glycosidase, 0.306 µg/mL on α-amylase enzymes, and 1.985 µg/mL on AChE. Tacrine was used as a positive control for AChE inhibition with an IC 50 value of 0.597 nM against AChE [16].
The three-dimensional structure of human hCA II has been demonstrated by X-ray crystallography [79]. It contains a zinc ion (Zn 2+ ) along with residues Thr199, Glu106, and His64 that directly participate in the catalytic activity in the active site of this dominant and cytosolic isozyme. The His94 residue acts as a shuttle, transferring a proton from the zincbound water to the solvent medium. MEAA and WEAA were tested against cytosolic hCA II isoenzymes. According to Table 4, it is depicted that extracts inhibit the hCA II enzyme. hCA isozymes take part in some biochemical and physiological processes as well as playing an important role in some diseases, such as cerebral edema, obesity, cancer, glaucoma, altitude sickness, and epilepsy. Cytosolic hCA II is highly expressed in most organs and contributes to many important physiological processes. Recently, hCA inhibitors have been commonly used as novel antiglaucoma, diuretics, antiobesity, anticancer, anticonvulsant, and anti-infective medications [65].
In addition to metabolically providing CO 2 transfer, the hCA enzyme plays a role in the accumulation of H + and HCO 3 − in many tissues. The hCA II isoenzyme is one of the most effective enzymes in erythrocytes and is found in almost every tissue and organ, including the eye, cornea, ciliary epithelium, kidney, central nervous system, and inner ear [80]. It was claimed that hCA II causes glaucoma and impaired vision by increasing HCO 3 − secretion in the eye's anterior uvea. They are involved in the secretion of bicarbonate from the exocrine glands in the digestive system [81]. They play a role in regulating the acidity of gastric juice and in the secretion of mucus and bicarbonate from epithelial cells on tissue surfaces in the gastrointestinal tract. In addition, the hCA II isoenzyme is effective in adjusting the intracellular pH and Ca 2+ level to prevent bone resorption [82]. Most hCA I is found in erythrocytes. However, its activity is only 15% of the hCA II isoform. Of all the CAs, CA II has the largest cytosolic distribution and is an isozyme with high activity [83]. Many phenolic acids and phenolic natural products, such as p-hydroxybenzoic acid, pcoumaric acid, ellagic acid, caffeic acid, ferulic acid, gallic acid, tannic acid, syringic acid, quercetin, ellagic acid, etc., have the ability to inhibit carbonic anhydrase. To the best of our knowledge, carbonic anhydrase enzyme inhibition has been tested for the first time on Astragalus species. Thus, the gap in the literature has been filled.

Plant Material
The aerial parts of A. alopecurus were collected by Dr. Leyla Güven from Erzurum Köşk village, 40 • 6 13" K, 41 • 24 32" D, at 1890 m altitude, on 1 July 2018. A. alopecurus was diagnosed by Prof. Dr. Yusuf Kaya from the Atatürk University Science Faculty. The herbarium specimens have been conserved at the Biodiversity Application and Research Center of Atatürk University with the AUEF 1395 herbarium number.

Preparation of Extracts (MEAA and WEAA)
For the preparation of MEAA, 50 g of aerial parts of the plant were crushed and extracted with 500 mL of methanol at room temperature using a mechanical stirrer [84]. The resulting mixture was filtered through Whatman No. 1 paper and concentrated until the rotary evaporator (Heidolph VV2000, Schwabach, Germany) was completely free of methanol. The resulting extract was stored at −18 • C until the study was carried out [85].
To prepare WEAA of the above-ground part of the plant, 500 mL of boiled water was poured onto the 30 g powdered plant, stirred in a magnetic stirrer for 1 h, and filtered with Whatman No. 1 paper [86]. The extract was evaporated in a rotary evaporator (Heidolph VV2000 Schwabach, Germany) and lyophilized. The lyophilized extract was stored at −18 • C until the study was carried out. The yields of methanol and water extracts from the aerial parts of the plant are 23.64 and 20.60% (w/w), respectively [87].

Determination of Total Phenolic and Flavonoid Contents
The total phenol content in MEAA and WEAA was determined by a spectrophotometric method based on the color reaction of phenolic compounds with the Folin-Ciocalteu reagent [88], as described in a previous study [89]. A spectrophotometric method based on the color reaction of flavonoids with aluminum chloride and potassium acetate was used to determine the total flavonoid content in plant extracts [90]. The quantity of total phenolics and flavonoids in extracts of A. alopecurus was determined as gallic acid equivalent (GAE) and quercetin equivalent (QE) from the equations obtained from the graphics of standards [91].

LC-MS/MS Instrumentation and Chromatographic Conditions
The phenolic composition in MEAA was detected by an Agilent Technologies 1290 Infinity UPLC chromatography equipped with an Agilent 6460 Triple Quadrupole mass spectrometer (Agilent Technologies, Palo Alto, Santa Clara, CA, USA) equipped with an electrospray ionization (ESI) source operating in negative multiple reaction monitoring (MRM) modes [92]. Chromatographic separation was performed on a Zorbax SB-C18 (4.6 × 100 mm, 3.5 µm) column at 30 • C by using a mobile phase consisting of water containing 0.1% formic acid (A) and acetonitrile containing 0.1% formic acid (B). The chromatography was performed by gradient elution. The gradient profile (time, % B) set was as follows: 0-4 min, 5% B; 4-7 min, 20% B; 7-14 min, 90% B; 15 min, 90% B; 15-15.1 min, 5% B; 15.1-17 min, 5% B at a 0.4 mL/min flow rate. An aliquot (5 µL) of the sample was injected, and the total run time was 17 min. The mass spectrometry conditions were set with a nitrogen gas temperature of 350 • C with a flow rate of 12 L/min, a sheath gas temperature of 250 • C with a flow rate of 5 L/min, and a nebulizer gas pressure of 55 psi. A complete mass scan ranging from 50 to 1300 m/z and the Agilent MassHunter Workstation to complete data acquisition and analysis were used.

Fe 3+ Reducing Assay
The reducing capacity of MEAA and WEAA was assessed using the Fe 3+ reducing technique, which differed from the FRAP and CUPRAC procedures [93], as given in the details [94]. The direct reduction in Fe 3+ (CN − ) 6 identified the decreasing quantity in this manner. Then, by adding excess ferric ions (Fe 3+ ), the Perls' Prussian blue complex was formed [95]. First, 0.75 mL of MEAA and WEAA with varying concentrations (15-45 g/mL) were mixed with K 3 Fe(CN) 6 (1%, 1.25 mL) and buffer (1.25 mL, 0.2 M, pH 6.6) solutions. The mixture was then incubated for 30 min at 50 • C. Next, the mixture was treated with 1.25 mL of trichloroacetic acid (TCA, 10%) and 0.5 mL of FeCl 3 (0.1%) before the absorbance was measured at 700 nm [96].

FRAP Reducing Assay
The FRAP approach is based on the acidic reduction in the Fe 3+ -TPTZ combination. At 593 nm, the enhanced absorbance was detected [97], as given in prior studies [98]. A fresh TPTZ solution (10 mM) was made and combined with buffer solution (pH 3.6, 0.3 M) and a 20 mM FeCl 3 solution in water for this purpose. Different amounts of MEAA and WEAA (15-45 µg/mL) were dissolved in 5 mL of suitable buffer, mixed, and kept at 25 • C for 30 min. Finally, absorbances at 593 nm were measured [99].

Cu 2+ Reducing Assay
The CUPRAC test was used to assess the reducing capabilities of MEAA and WEAA [100], as given in a prior study [101]. Neocuproine was utilized as a chromogenic oxidizing agent in this approach [102]. To begin, 1 mL of acetate buffer (1.0 M), neocuproine (7.5 mM), and CuCl 2 solution (10 mM) were added to each tube and vortexed. All samples were put into tubes at concentrations ranging from 15 to 45 µg/mL. With distilled water, the tubes were filled to 1 mL. The samples were maintained at 25 • C for 30 min, and the absorbance was recorded at 450 nm [103].

DPPH Radical Scavenging Assay
Using the DPPH scavenging technique, the free radical scavenging capacity of MEAA and WEAA was assessed [103]. The technique relies on antioxidants to remove DPPH free radicals. Standards and extracts were generated with concentrations ranging from 15 to 45 µg/mL. For each sample, 500 µL of DPPH (0.1 mM) was added to tubes. For 30 min, these tubes were kept in the dark at 25 • C. At 517 nm, the measurements were taken. Samples of DPPH potentials were calculated and compared to standards. Finally, the IC 50 values for each sample were determined. The decrease in absorbance demonstrates the sample's capacity to scavenge DPPH free radicals [104].

ABTS Radical Scavenging Assay
A second radical scavenging technique, the ABTS •+ scavenging test [104], was employed to assess MEAA and WEAA's capacity to scavenge free radicals [105]. First, an ABTS radical cation was produced, per this experiment, and then K 2 S 2 O 8 (2.45 mM) and ABTS (7.0 mM) interacted [106]. Prior to measurement, the solution's absorbance was corrected with buffer solution to 0.750 ± 0.025 at 734 nm (pH 7.4, 0.1 M). Then, MEAA and WEAA at varying concentrations (15-45 µg/mL) were added to 1 mL of ABTS •+ solution. ABTS •+ scavenging of all samples was assessed at 734 nm after 30 min. The decrease in absorbance demonstrates the sample's capacity for free radical scavenging when treated with ABTS •+ [107].

DMPD Radical Scavenging Assay
The DMPD •+ scavenging potential of MEAA and WEAA was determined using a slightly modified approach described previously by Fogliano et al. [108] and a prior study [109]. For this purpose, 200 µL of 0.05 M FeCl 3 and 1 mL of DMPD solution were added to the 0.1 M buffer (100 mL, pH 5.3). All samples were produced with concentrations ranging from 15 to 45 µg/mL. Water was used to reduce the total volume to 0.5 mL. After an hour of incubation, 1 mL of DMPD •+ solution was transferred, and absorbance at 505 nm was measured prior to the studies [110].

Metal Chelating Assay
The Fe 2+ chelating effect of MEAA and WEAA was carried out as described in the literature by Re et al. [111] and previous studies [112]. Different concentrations (15-45 µg/mL) of extracts and standard compounds were transferred to a 0.125 mL FeSO 4 (2 mM) solution. In this way, Fe 2+ ions interact with the phenolic compounds in the extract, and Fe 2+ ions are chelated by the sample. Next, 0.5 mL of Tris-HCl solution (pH 7.4) is added and incubated for 30 min. Then, 0.75 mL of 0.2% bipyridyl solution dissolved in HCl (0.2 M), 0.125 mL of ethanol, and 0.595 mL of water are added to the mixture, respectively, and incubated for 15 min. Ethanol was used as a blank, and absorbances were measured at 522 nm [113].

Antidiabetic Assay
The ability of methanol and water extracts to inhibit α-glycosidase and α-amylase enzymes was examined to establish the plant's potential as an antidiabetic. A p-Nitrophenyl-D-glycopyranoside (p-NPG) substrate, as described in a previous study [117], was used to test the inhibitory effectiveness of MEAA and WEAA on α-glycosidase and α-amylase enzymes. At first, α-glycosidase enzyme solution (10-30 µL, 0.15 U/mL), the sample (10-50 µL), and 50 µL of p-NPG (5 mM) were mixed and incubated for 3 min at room temperature. The absorbance of the mixture was monitored at 405 nm prior to the study [118].
For the α-amylase enzyme inhibition assay, it was performed according to our previous study [119]. First, 2 g of starch was dissolved in a 100 mL solution of 0.4 M NaOH before being heated at 80 • C for 15 min. The pH was changed to 6.9 after cooling, and distilled water was used to make the total volume 100 mL. Then, 5 µL of extracts were added to 35 µL of phosphate buffer (pH 6.9) and 35 µL of starch solution. Next, 20 µL of the enzyme solution was added, and the mixture was again incubated at 25 • C for 20 min. The reaction was finished by adding 50 µL of HCl (0.1 M). At a wavelength of 580 nm, the mixture's absorbance was measured prior to the studies [120].

Antiglaucoma Assay
In order to examine the inhibitory effects of methanol and water extracts of A. alopecurus aerial parts on the hCA II isoform, this isoform was purified by Sepharose-4B-L-Tyrosinesulfanilamide affinity chromatography from human red blood cells [121]. Human erythrocyte samples were centrifuged for 30 min at 13,000 rpm for this reason. After that, the solution was filtered. At pH 8.7, solid Tris was added to the serum to isolate the hCA II isoenzyme. With buffer solution (pH 8.7, 25 mM Tris-HCl/0.1 M Na 2 SO 4 ), the affinity column was adjusted. The hCA II isoenzyme was cleaned with the buffer solution (pH 5.6, 0.1 M sodium acetate/0.5 M NaClO 4 ). The hCA II isoenzyme was fractionated from the column into Eppendorf tubes (2 mL). All investigations were conducted at 4 • C. A Thermo Scientific brand spectrophotometer was used to monitor the change in absorbance of the p-nitrophenolate ion of p-nitrophenylacetate for 3 min at room temperature in order to determine the activity of the hCA II isoenzyme [122]. The measured test tube has the following contents: 0.1 mL of enzyme solution, 0.2 mL of water, and 0.4 mL of 0.05 M Tris-SO 4 buffer (pH 7.4) [123]. The purity of the hCA isoform was controlled by the SDS-PAGE purity technique [124]. The protein quantity was determined at 595 nm according to the Bradford method [125], as given previously [126]. The spectrophotometric Verpoorte method (Shimadzu, UVmini-1240 UV-VIS) was employed for performing CA activity [73,127].

IC 50 Value Determination
The half maximal inhibition concentration (IC 50 ) values were calculated from activity (%) versus different concentrations of MEAA and WEAA [128].

Statistical Analysis
Statistical analyses were used to evaluate anticholinergic, antidiabetic, and antioxidant activity results by unpaired Student's t-test (GraphPad, La Jolla, CA, USA. Software 7.0). All results were given as means with their standard deviation (SD). p < 0.05 was taken as the minimum level of significance.

Conclusions
Our study provided important findings regarding the high concentration of isorhamnetin, fumaric acid, and rosmarinic acid in A. alopecurus with an LC-MS/MS analysis, and it was concluded that A. alopecurus can be used commercially due to its phenolic compounds, antioxidants, and enzyme inhibition abilities. A. alopecurus contains quantities of bioactive secondary metabolites, such as phenolic and flavonoid compounds. Furthermore, the A. alopecurus extract was found to be rich in phenolic contents, antioxidant ability, reducing power, AChE, α-amylase, α-glycosidase, and hCA II inhibition profiles. A. alopecurus can also be used as a natural source for the treatment of T2DM, AD, and glaucoma. From this perspective, inhibition studies on the AChE enzyme are planned to determine the anti-AD effects of A. alopecurus extracts. Additionally, the inhibition of the hCA II enzyme was analyzed to determine a link with glaucoma. Furthermore, the antidiabetic potential of A. alopecurus extracts has been realized by identifying α-amylase and α-glycosidase inhibition. Additionally, Cu 2+ , Fe 2+ , and Fe 3+ -TPTZ reducing, as well as DPPH and ABTS scavenging, tests were performed to understand the antioxidant ability of A. alopecurus extracts. Furthermore, total phenolics and flavonoids in A. alopecurus were determined for both extracts. A phenolic analysis was performed by LC-MS/MS to define the biological effects of the chemical profile of A. alopecurus. Although our current laboratory conditions are not suitable, we plan to conduct in vivo studies for this research in the future, which will be supported by experimental animals.